Metabolic engineering: Techniques for analysis of targets for genetic manipulations

Abstract

Metabolic engineering has been defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. With this definition metabolic engineering includes: (1) inserting new pathways in microorganisms with the aim of producing novel metabolites, e.g., production of polyketides by Streptomyces; (2) production of heterologous peptides, e.g., production of human insulin, erythropoitin, and tPA; and (3) improvement of both new and existing processes, e.g., production of antibiotics and industrial enzymes. Metabolic engineering is a multidisciplinary approach, which involves input from chemical engineers, molecular biologists, biochemists, physiologists, and analytical chemists. Obviously, molecular biology is central in the production of novel products, as well as in the improvement of existing processes. However, in the latter case, input from other disciplines is pivotal in order to target the genetic modifications; with the rapid developments in molecular biology, progress in the field is likely to be limited by procedures to identify the optimal genetic changes. Identification of the optimal genetic changes often requires a meticulous mapping of the cellular metabolism at different operating conditions, and the application of metabolic engineering to process optimization is, therefore, expected mainly to have an impact on the improvement of processes where yield, productivity, and titer are important design factors, i.e., in the production of metabolites and industrial enzymes. Despite the prospect of obtaining major improvement through metabolic engineering, this approach is, however, not expected to completely replace the classical approach to strain improvement—random mutagenesis followed by screening. Identification of the optimal genetic changes for improvement of a given process requires analysis of the underlying mechanisms, at best, at the molecular level. To reveal these mechanisms a number of different techniques may be applied: (1) detailed physiological studies, (2) metabolic flux analysis (MFA), (3) metabolic control analysis (MCA), (4) thermodynamic analysis of pathways, and (5) kinetic modeling. In this article, these different techniques are discussed and their applications to the analysis of different processes are illustrated. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:125–132, 1998.

Control of the threonine-synthesis pathway in Escherichia coli: a theoretical and experimental approach

“…The goal of metabolic engineering is the rational design and implementation of changes in an organism's properties to alter its metabolism in the desired manner [1]. One particular area of interest is the possibility of increasing rates and yields of amino acid synthesis by micro-organisms.…”

“…If this enzyme is over-expressed, then inevitably its flux control coefficient falls and the flux control coefficient of one or more of the other enzymes rises, so that another point in the pathway becomes a more significant focus for further action. A theoretical method for identifying a set of linked changes on the basis of control 1 To whom correspondence should be addressed (e-mail JP.Mazat!phys-mito.u-bordeaux2.fr).…”

Biological network analyses: computational genomics and systems approaches

“…The quantitative analysis of metabolic networks and other metabolic engineering techniques have traditionally been applied to optimize cellular function primarily for the purpose of enhancing the production of specialty chemicals from prokaryotic cells, yeasts, fungi and to some limited extent, mammalian cells [44,45]. However, prior to the application of metabolic engineering techniques, the network map of the metabolic pathways for the cell or tissue of interest must be constructed.…”

The role of membrane transport in metabolic engineering of plant primary metabolism

“…This includes adjustment of the amount and kinetic properties of metabolic enzymes as well as the regulatory networks that govern the expression of pathway components, which in most cases is achieved through genetic engineering [1,2]. Thanks to the development of mathematical models of metabolism, the concepts of metabolic engineering have been successfully applied to the engineering of microbial metabolism, for example to increase the amount of desired amino acids or fermentation products [3], as well as the production of plant specialized metabolites in yeast, such as artemisinic acid [4].…”